Abstract
Context: Curcumin is a polyphenolic compound extracted from rhizomes of the tropical plant Curcuma longa L. (Zingiberaceae) and it has antitumor, antioxidative, and anti-inflammatory effects. However, its effects on leukemia cell proliferation and invasion are not clear.
Objective: This study investigates the effects of curcumin on acute monocytic leukemia SHI-1 cells at the molecular level.
Materials and methods: The effects of SHI-1 cells treated with 6.25–25 μM curcumin for 12–48 h were measured by MTT assay, flow cytometry, and Matrigel transwell assay; the underlying molecular mechanisms were assessed by quantitative PCR, Western blotting, and gelatin zymography.
Results: Treatment of SHI-1 cells with curcumin inhibited cell proliferation in a dose- and time-dependent manner, and the IC50 values at 12, 24, and 48 h were 32.40, 14.13, and 9.67 μM. Curcumin inhibited SHI-1 cell proliferation by arresting the cells in the S-phase, increasing the number of Annexin V-FITC+/PI− cells and promoting the loss of △Ψm. The results of PCR and Western blotting showed that curcumin increased the FasL mRNA level; inhibited Bcl-2, NF-κB, and ERK expression; and activated P38 MAPK, JNK, and caspase-3. Additionally, curcumin partially suppressed SHI-1 cell invasion and attenuated the mRNA transcription and secretion of MMP-2 and MMP-9.
Discussion and conclusion: This study demonstrates that curcumin not only induces SHI-1 cell apoptosis, possibly via both intrinsic and extrinsic pathways triggered by JNK, P38 MAPK and ERK signaling, but also partially suppresses SHI-1 cell invasion, likely by reducing the levels of transcription and secretion of MMP-2 and MMP-9.
Introduction
Acute monocytic leukemia is a common subtype of acute myeloid leukemia (AML) with characteristic clinical features such as hyperleukocytosis, extramedullary involvement, and chromosomal abnormalities in karyotype (Nakamaki & Tsuruoka, Citation1998). The clinical complete remission rates of acute monocytic leukemia are relatively low, and a considerable number of patients die due to chemotherapy drug resistance as well as intramedullary and extramedullary relapse. In recent years, researchers have made great progress in understanding the pathogenesis and treatment of AML; however, specific targeted medicines for acute monocytic leukemia are still lacking.
Curcumin, a polyphenolic compound extracted from the rhizome of the tropical plant Curcuma longa L. (Zingiberaceae), has antitumor, antioxidative, anti-inflammatory, and hypolipidemic effects (Gopal et al., Citation2014). The American National Cancer Institute has ranked curcumin as a third-generation cancer chemopreventive drug (Thiyagarajan et al., Citation2009). Curcumin regulates the expression of pro- and anti-apoptotic proteins, such as p53, Bcl-2, Mcl-1, NF-κB, caspases, and mitogen-activated protein kinases (MAPKs), and it has been found to mainly inhibit proliferation and induce apoptosis of various types of solid tumors and leukemia cell lines (Kelkel et al., Citation2010; Reuter et al., Citation2008). With respect to AML, curcumin exerts inhibitory effects on MDR1, WT1, and leukemic cell proliferation in patients (Gao et al., Citation2012). Moreover, curcumin induces apoptosis in the HL-60 cell line through the ornithine decarboxylase-dependent and endoplasmic reticulum stress pathways as well as through inhibition of telomerase activity (Liao et al., Citation2008). Curcumin can also inhibit the invasion and metastasis of certain solid tumor cells (Xu et al., Citation2014). For example, curcumin inhibits TGF-β1-induced matrix metalloproteinase (MMP)-9 expression and invasion in breast cancer MDA-MB-231 cells through ERK and Smad signaling (Mo et al., Citation2012). Curcumin also suppresses lung cancer cell migration and invasion through the Rac1-dependent signaling pathway (Chen et al., Citation2014). However, the effects of curcumin on invasion and metastasis in leukemia cells have remained unknown until now.
SHI-1 is an acute monocytic leukemia cell line that was originally derived from mononuclear cells from the bone marrow of a 37-year-old male with M5b in relapse and has been maintained as a stable cell line in vitro (Chen et al., Citation2005). SHI-1 cells have an abnormal t (6; 11) (q27; q23) translocation and express the pathogenic fusion product of MLL/AF6. Moreover, SHI-1 cells also have a mutation in the p53 gene and show high tumorigenicity, high invasive ability, and drug resistance in nu/nu mice (Chen et al., Citation2007). In the present study, for the first time, we find that curcumin not only significantly induces apoptosis but also partially suppresses invasion in SHI-1 cells in vitro. The results provide a theoretical basis for future basic research on and clinical treatment of acute leukemia.
Materials and methods
Cells and main reagents
The SHI-1 cell line was a kind gift from the Jiangsu Institute of Hematology. SHI-1 cells were incubated with IMDM medium (Gibco, Grand Island, NY) containing 15% fetal bovine serum (Gibco, Grand Island, NY) at 37 °C in a 5% CO2 incubator. Curcumin (purity ≥ 94%), DMSO, MTT, PI, JC-1, and gelatin were purchased from Sigma-Aldrich (St. Louis, MO). The caspase-3 detection kit was purchased from BioVision (Milpitas, CA). Monoclonal antibodies to anti-human MAPK, phospho-MAPK, p53, and NF-κB were purchased from Cell Signaling Technology (Danvers, MA). TRIzol, the RNA reverse transcription kit and SYBR Green fluorescent quantitative PCR mix were purchased from Takara Biotechnology (Dalian, China).
Cytotoxicity assay
SHI-1 cells in the logarithmic growth phase were collected and seeded in 96-well plates at 5.0 × 103 cells/well. After 2 h, 6.25, 12.5, and 25 μM curcumin were added to the cells for another 12, 24, or 48 h. Simultaneously, a solvent control group treated with 0.75% DMSO, a normal cell group without any drugs added, and a blank control group without any cells were established. Three hours before collection, 20 μl of MTT solution was added to each well. Formazan crystals were formed, and the percentage of viable cells was determined by absorbance measurements at 490 nm with an ELISA reader.
DAPI staining
To visualize apoptosis, SHI-1 cells treated with or without curcumin for 24 h were stained with DAPI. Briefly, after treatment with different concentrations of curcumin, the cells were fixed with 4% paraformaldehyde prior to washing with PBS. The washed cells were then stained with 2 μg/ml DAPI for 15 min in the dark. Cell nuclei were observed via laser scanning confocal microscopy at 340–380 nm.
Measurements of cell cycle, early apoptosis and ▵Ψm
For cell-cycle analysis, SHI-1 cells that had been treated with 6.5, 12.5, and 25 μM curcumin for 24 h were harvested, washed with cold PBS and fixed with 70% ethanol at 4 °C overnight. After washing with PBS, the cells were incubated with RNase A for 5 min. After incubation with PI (200 μg/ml), the cells were analyzed by flow cytometry (FCM; Beckman, Krefeld, Germany).
To detect early apoptosis, 100 μl of the SHI-1 cell suspension (1.0 × 105 cells) was mixed with 5 μl of FITC-Annexin V and 2 μl of PI in a cultivation tube. The tubes were protected from light and gently shaken for 15 min before adding 400 μl of 1 × binding buffer to each tube. Cell apoptosis in the early stage was quantitatively detected by FCM.
ΔΨm was determined using the dual-emission potential-sensitive probe JC-1. SHI-1 cells from all the groups were collected and incubated with 10 μM JC-1 for 20 min at 37 °C in the dark. The cells were then washed twice with PBS and analyzed by FCM.
Caspase-3 activity assay
SHI-1 cells that were or were not treated with varying concentrations of curcumin for 24 h were washed twice with ice-cold PBS; then, cell lysates were prepared, and protein concentrations were estimated. The lysates (100 μg of protein in 50 μl of lysis buffer) were combined with 50 μl of 2 × reaction buffer (containing 10 mM DTT) and the caspase-3 substrate Ac-DEVD-para-nitroanilide (pNA) (4 mM, 5 ml); they were then incubated at 37 °C for 2 h. The release of the chromophore pNA was quantified by measuring the absorbance at 405 nm with an ELISA reader.
Real-time quantitative PCR
Using Trizol reagent, total RNA was extracted from SHI-1 cells that were or were not treated with curcumin for 24 h. All RNA samples were reverse-transcribed individually using a reverse transcription kit according to the instructions of the manufacturer. PCR amplification was performed in a volume of 20 μl using SYBR Green fluorescent quantitative PCR mix (Takara, Dalian, China). Gene-specific PCR primers were synthesized by Shanghai Sangon Biotech (). Quantitative PCR amplification of each sample was performed via the following protocol: 40 cycles of 95 °C for 35 s, 60 °C for 30 s, and 72 °C for 15 s. Each assay was analyzed on a Roche Light Cycler PCR system in triplicate, and the fold change in expression was determined using the comparative CT method.
Table 1. PCR primer sequences.
Western blot analysis
SHI-1 cells that did or did not undergo treatment with curcumin for 48 h were collected and lysed in RIPA buffer. Nuclear and cytoplasmic proteins were extracted using a kit (Beyotime, Shanghai, China). The cytoplasmic proteins were prepared via repeated freeze/thaw cycles in cold buffer A and buffer B, and the nuclear proteins were extracted using ice-cold buffer C according to the manual provided with the kit. Equal amounts of extracted proteins were denatured and subjected to SDS-PAGE. After electrophoresis and membrane transfer, PVDF membranes (Millipore, Billerica, MA) containing the proteins were blocked with 5% BSA at room temperature (RT) for 2 h and then incubated with the primary antibody at 4 °C overnight. The membranes were washed three times in TBST and subsequently probed with HRP-conjugated goat anti-mouse IgG at RT for 2 h. After incubation, the blots were washed, and ECL reagents were added. Detection was performed using an ImageQuant LAS 4000 mini (GE, Louisville, KY).
Gelatin zymography
SHI-1 cells were incubated in serum-free medium with or without curcumin for 24 h. The supernatants were collected, and protein concentrations were measured. Each supernatant sample containing 40 μg of protein was run on 10% SDS-PAGE gels copolymerized with 0.1% gelatin at 4 °C. After electrophoresis, the gels were washed three times with renaturing buffer containing 2.5% Triton X-100 (v/v) for 30 min and briefly rinsed with washing buffer. Then, the gels were incubated at 37 °C for 42 h in developing buffer containing 0.02% Brij-35. The gels were subsequently stained with 0.25% Coomassie brilliant blue (G250) at RT for 3 h and destained with a solution containing 10% acetic acid and 20% methanol. Enzyme-digested regions were visualized as light bands against a blue background.
Invasion assay
The Costar transwell chamber system was used to measure cell invasion. SHI-1 cells that were either treated or not with low concentrations of curcumin at 4, 6, and 8 μM for 24 h were suspended in 200 μl of serum-free IMDM and seeded at a final density of 2.0 × 105 cells/well in the upper chamber in triplicate. The lower compartment of the invasion chamber was filled with 800 μl of IMDM (supplemented with 10% human serum) containing 100 ng/ml SDF-1α as a chemoattractant. The chambers were incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The invasion rate was calculated based on the ratio of the number of SHI-1 cells in the medium of the lower compartment to the total number of cells (2.0 × 105 cells) loaded into the upper compartment.
Statistical analyses
All experiments were repeated at least three times, and the data are expressed as the mean ± standard deviation (SD). The statistical software SPSS 15.0 (SPSS Inc., Chicago, IL) was used for assessment. Student's t-test was used to compare means of two groups, and one-way ANOVA was used to compare means of multiple samples; p < 0.05 was considered significant.
Results
Curcumin inhibits SHI-1 cell proliferation
Cytotoxicity was measured using the MTT assay. As shown in , curcumin inhibited SHI-1 cell proliferation in a dose- and time-dependent manner. The rates of proliferation inhibition did not significantly differ between the 0.75% DMSO solvent control group and the normal cell group (p > 0.05), suggesting that 0.75% DMSO did not affect this assay. However, compared with the normal group, SHI-1 cells incubated with 6.25, 12.5, and 25 μM curcumin for different durations had significantly different proliferation rates (p < 0.01). In addition, the IC50 values of SHI-1 cells treated with curcumin for 12, 24, and 48 h were 32.40, 14.13, and 9.67 μM, respectively.
Figure 1. Inhibitory effect of curcumin (Cur) on SHI-1 cell proliferation. Each point represents the mean ± SD of three independent experiments. The inhibitory effects of curcumin (6.5, 12.5, and 25 μM) and 0.75% DMSO were determined using an MTT assay at 12, 24, and 48 h. The results suggest that 0.75% DMSO (solvent) had no effect on cell viability. Compared with the proliferation of cells in the normal control group, the proliferation of SHI-1 cells treated with different concentrations of curcumin was significantly inhibited (p < 0.01).
Curcumin augments nuclear fragmentation in SHI-1 cells
As revealed via DAPI staining, the nuclei of untreated normal SHI-1 cells were regular and small, whereas those of the cells exposed to curcumin for 24 h were condensed and enlarged. The observed changes in SHI-1 cell nuclei occurred in a dose-dependent manner. Of note, the nuclei of certain SHI-1 cells were fragmented and formed falcate apoptotic bodies in the group treated with 25 μM curcumin (). This result shows that curcumin might induce apoptosis in SHI-1 cells and thus inhibit their proliferation.
Figure 2. The curcumin-induced changes in the nuclear morphology of SHI-1 cells were imaged and analyzed by laser scanning confocal microscopy (magnification, × 400). In the control group, the normal SHI-1 cells were dense, with nuclei that were small and regular. In contrast, the SHI-1 cells in the curcumin groups were increasingly sparse, with condensed nuclei. Curcumin exerted dose-dependent effects on these cells. In the group treated with 25 μM curcumin, certain nuclei fragmented and formed falcate apoptotic bodies (as marked).
Curcumin induces SHI-1 cell apoptosis by S phase arrest and loss of ΔΨm
To further investigate the influence of curcumin on apoptosis, FCM was used to assess the cell cycle, early apoptosis, and △Ψm of normal control SHI-1 cells and those treated with 6.25, 12.5, and 2 μM curcumin for 24 h. As shown in , curcumin treatment caused a significant increase in the S phase fraction of SHI-1 cells, while the fraction of cells in the G1 phase decreased in a dose-dependent manner compared with normal control SHI-1 cells. In particular, there was an obvious sub-G1 apoptotic peak in the 25 μM curcumin group. This result suggests that curcumin affects SHI-1 cell proliferation by inducing S-phase arrest.
Figure 3. Analysis of the cell cycle of SHI-1 cells treated with different concentrations of curcumin by FCM. SHI-1 cells treated with different concentrations of curcumin were arrested in S phase in a dose-dependent manner, and there was a clear sub-G1 apoptotic peak in the group treated with 25 μM curcumin.
The Annexin V-FITC+/PI− cells detected by FCM are those that are undergoing early apoptosis. As shown in , the number of Annexin V-FITC+/PI− SHI-1 cells in the curcumin groups increased in a dose-dependent manner compared with that of the control group (p < 0.01). Additionally, the loss of △Ψm is also an event observed in early apoptosis. The percentage of normal △Ψm cells in curcumin-treated groups was decreased (p < 0.01), indicating that curcumin promoted a dose-dependent loss of △Ψm in SHI-1 cells. Thus, curcumin induced early apoptosis in SHI-1 cells, likely in a manner that involves the intrinsic mitochondrial pathway.
Table 2. Rates of early apoptosis (Annexin V-FITC+/PI−) and △Ψm values of SHI-1 cells treated with curcumin at different concentrations.
Curcumin induces SHI-1 cell apoptosis via the caspase-dependent pathway
Preliminary experiments indicated that caspase-3 activity in SHI-1 cells first exponentially increased up to 2 h and plateaued thereafter. Therefore, lysates were prepared from SHI-1 cells that were either treated with curcumin or untreated for 24 h, incubated with Ac-DEVD-pNA at 37 °C for another 2 h and detected with an ELISA reader. As shown in , curcumin elevated caspase-3 activity in a dose-dependent manner. Because caspase-3 is a core effector molecule of the apoptotic pathway mediated by caspases, we inferred that curcumin likely induced apoptosis in SHI-1 cells via a caspase-dependent pathway.
Figure 4. Effect of curcumin on caspase-3 activity in SHI-1 cells. The caspase-3 activity of SHI-1 cells was determined in three independent experiments and is presented as the mean ± SD. Curcumin at 6.25 μM did not significantly alter the caspase-3 activity; however, 12.5 μM and 25 μM concentrations significantly increased caspase-3 activity in SHI-1 cells. Note: *p < 0.01 versus the control group (normal SHI-1 cells); #p < 0.05 versus the control group.
Curcumin induces SHI-1 cell apoptosis by modulating the expression of Bcl-2, FasL, and MMP mRNA
Real-time quantitative PCR showed that curcumin treatment not only significantly decreased Bcl-2, MMP-2, and MMP-9 mRNA expression but also increased FasL mRNA expression in a dose-dependent manner (). However, curcumin failed to affect the mRNA expression of the apoptosis-related gene survivin and the pathogenic fusion gene MLL-AF6 in SHI-1 cells.
Figure 5. Effect of curcumin on Bcl-2, FasL, surviving, and MMP mRNA expression in SHI-1 cells. The 2−△△Ct value of each gene was calculated, indicating the relative mRNA expression in SHI-1 cells induced by exposure to 6.25–25 μM curcumin for 24 h. The results showed that curcumin down-regulated Bcl-2, MMP-2, and MMP-9 mRNA; up-regulated FasL mRNA; and had no effect on survivin and MLL-AF6 genes in SHI-1 cells. Note: *p < 0.01 versus the control group (normal SHI-1 cells); #p < 0.05 versus the control group.
FasL is involved in the extrinsic apoptotic pathway, and the Bcl-2 family also plays a vital role in cell apoptosis. It was previously reported that curcumin inhibited the expression of Bcl-2 and coordinately activated caspase-3, eventually leading to apoptosis of the Kasumi-1 and KG1a leukemia cell lines (Akl et al., Citation2014). Moreover, Moreover MMP-2 and MMP-9 gelatinases play a major role in extracellular matrix degradation and remodeling, which is closely related to tumor cell invasion and metastasis (Klein et al., Citation2004). Therefore, we deduced that curcumin not only induces SHI-1 cell apoptosis through changes in the mRNA expression of FasL and Bcl-2 but also likely inhibits SHI-1 cell invasion and metastasis by inhibiting the mRNA expression of MMP-2 and MMP-9.
Curcumin induces SHI-1 cell apoptosis by activating MAPK and inhibiting NF-κB
In human cells, the MAPK family comprises three main members: P38 MAPK, JNK, and ERK. These proteins regulate cell proliferation, apoptosis, and differentiation. Thus, we next examined whether MAPK family members were involved in the curcumin-mediated apoptosis of SHI-1 cells. In addition to increasing the phosphorylation levels of P38 MAPK and JNK, curcumin also decreased the phosphorylation of ERK in a dose-dependent manner. Compared with their levels in the normal control group, the expression levels of p-P38 MAPK, p-JNK, and p-ERK in the 12.5 and 25 μM curcumin groups significantly differed (p < 0.01, ). These data suggested that P38 MAPK, JNK, and ERK signaling pathways may all be involved in curcumin-induced SHI-1 cell apoptosis.
Figure 6. Effects of curcumin on P38 MAPK, JNK, ERK and NF-κB expression in SHI-1 cells. (A) SHI-1 cells were treated with 6.25–25 μM curcumin for 48 h, and the expression levels of MAPKs, p-MAPKs, and NF-κB proteins were detected by Western blot. (1) Control group (normal SHI-1 cells); (2) group treated with 6.25 μM curcumin; (3) group treated with 12.5 μM curcumin; (4) group treated with 25 μM curcumin. (B) The expression levels of MAPKs in SHI-1 cells treated with 6.25–25 μM curcumin were analyzed using p-P38 MAPK/P38 MAPK, p-JNK/JNK, and p-ERK/ERK, and the activity of nuclear NF-κB was analyzed by examining NF-κB/β-actin. The data are expressed as the mean ± SD of three independent experiments. Note: *p < 0.01 versus the control group (normal SHI-1 cells); #p < 0.05 versus the control group.
Additionally, we examined the protein expression of NF-κB and p53 in SHI-1 cells. As shown in , the expression of active NF-κB p65 protein in the nucleus of SHI-1 cells was reduced by curcumin treatment in a dose-dependent manner; however, p53 protein was not detected. Similarly, it has been reported that in SHI-1 cells, the p53 gene is mutated on one chromosome and is not present on the other chromosome (Chen et al., Citation2007). Therefore, curcumin-induced apoptosis of SHI-1 cells was likely also related to the inhibition of NF-κB activity.
Curcumin reduces the gelatinase levels of MMP-2 and MMP-9 in the culture supernatant of SHI-1 cells
The culture supernatant of SHI-1 cells incubated with or without curcumin was collected, and the gelatinase level was analyzed by gelatin zymography. There were two white bands in the gelatin, which were identified as proMMP-2 (72 kD) and proMMP-9 (92 kD; ). Scanning and analysis by Photoshop showed that the gelatinase levels of MMP-9 and MMP-2 were decreased in the curcumin groups in a dose-dependent manner, and the levels of the 12.5 and 25 μM curcumin groups were significantly different from that of the normal control group (p < 0.05, ). Combining these results with the real-time quantitative PCR results, we postulated that curcumin likely regulates the metastasis and invasion of SHI-1 cells by suppressing the mRNA transcription and gelatinase levels of MMP-2 and MMP-9.
Figure 7. Effect of curcumin on MMP-9 and MMP-2 gelatinase levels in the culture supernatant of SHI-1 cells. (A) SHI-1 cells were treated with 6.25–25 μM curcumin for 24 h, and the gelatinase levels of MMP-2 (72 kD, proMMP-2) and MMP-9 (92 kD, proMMP-9) in the culture supernatant were detected by a gelatin zymography assay. (1) Control group (normal SHI-1 cells); (2) group treated with 6.25 μM curcumin; (3) group treated with 12.5 μM curcumin; (4) group treated with 25 μM curcumin. (B) After scanning and analyzing the results in Photoshop, it was clear that the gelatinase levels of MMP-9 and MMP-2 were decreased in the curcumin groups in a dose-dependent manner. The groups treated with 12.5 and 25 μM curcumin exhibited significant differences compared with the control group. Note: *p < 0.01 versus the control group (normal SHI-1 cells); #p < 0.05 versus the control group.
Low-concentration of curcumin suppresses SHI-1 cell invasion
As mentioned above, 6.25 μM curcumin was less cytotoxic to SHI-1 cells after 24 h incubation. Furthermore, the mRNA levels and enzymatic activities of MMP-2 and MMP-9 were decreased by curcumin. Thus, we used low concentrations of curcumin at 4–8 μM to determine whether curcumin could suppress SHI-1 cell invasion in a Matrigel transwell assay. The results showed that the invasion rate of normal SHI-1 cells that were not treated with curcumin was 33.43 ± 1.91%, and the invasion rates in the groups treated with 6 and 8 μM curcumin were 26.08 ± 2.98% and 19.83 ± 3.01%, respectively. The invasion rate of the group treated with 8 µM curcumin was significantly different from that of the normal control group (p < 0.05). Therefore, we confirmed that curcumin not only induces SHI-1 cell apoptosis but also moderately suppresses SHI-1 cell invasion.
Discussion
Numerous studies have revealed that cell apoptosis occurs via two basic pathways: the death receptor-mediated extrinsic pathway and the intracellular mitochondrial-mediated intrinsic pathway. The former begins with the interaction of Fas and FasL. Subsequently, the p53, Bcl-2, and NF-κB pathways are activated, eventually leading to caspase-8 activation (Susin et al., Citation1999). The latter affects the ΔΨm, leading to cytochrome C release and an increase in the intracellular Ca2+ concentration, eventually leading to caspase-9 activation (MacEwan, Citation2002). The extrinsic and intrinsic apoptosis pathways both ultimately lead to activation of caspase-3, which acts as an endonuclease that promotes DNA strand breakage and causes disintegration of cellular structures (Fan et al., Citation2005).
MAPKs are serine/threonine protein kinases that play an important role in both extrinsic and intrinsic signal transduction pathways and induce the apoptosis of various cancer cells (Kim & Choi, Citation2010). P38 MAPK, JNK, and ERK are the three main members of the MAPK family in human cells. Generally, activation of the P38 MAPK and JNK pathways induces apoptosis, whereas ERK activation is often associated with cell survival (Zhu et al., Citation2013). Studies have shown that activated JNK not only increases the expression of P53, Bax, and FasL by enhancing AP-1 activity to mediate extrinsic apoptosis but also promotes the release of cytochrome C from the mitochondria through Bcl-2 and Bcl-xL phosphorylation to drive intrinsic apoptosis (Wagner & Nebreda, Citation2009). Similarly, activated P38 MAPK enhances the expression of c-myc and the phosphorylation of p53, and it is involved in extrinsic apoptosis mediated by Fas/FasL. Meanwhile, P38 MAPK regulates the expression of several transcription factors including NF-κB, HSP, and DNA damage factor (Bradham & McClay, Citation2006; Gasparini et al., Citation2014). However, ERK is commonly activated by the classic Raf/MEK/ERK signaling pathway to repress apoptosis. Activated ERK can up-regulate the expression of Bcl-2, activate the transcription factors Elk-1 and ATF2 and regulate the cell cycle to promote proliferation (Whelan et al., Citation2012).
In the current study, we found that treatment with 6.25–25 μM curcumin inhibited SHI-1 cell proliferation by arresting the cell cycle in S phase and distinctly induced SHI-1 apoptosis in a dose-dependent manner. Additionally, we found that curcumin promoted the loss of △Ψm; the activation of caspase-3, JNK, and P38 MAPK; the up-regulation of FasL mRNA expression; the inhibition of ERK and NF-κ B activities; and the reduction of Bcl-2 mRNA expression during SHI-1 cell apoptosis. Moreover, we discovered that curcumin did not affect the mRNA expression of the specific pathogenic fusion gene MLL-AF6 or the anti-apoptosis survivin gene in SHI-1 cells. At the same time, we confirmed that the wild type p53 gene is present in SHI-1 cells, which is consistent with work by Chen et al. (Citation2005). Combined with its effects on molecular mechanism of apoptosis, we hypothesized that curcumin might induce the activation of JNK and P38 MAPK to increase downstream FasL expression and inhibit downstream NF-κ B activity. This sequence of events would decrease Bcl-2 expression and promote the loss of △Ψm to activate both the intrinsic and extrinsic pathways, which ultimately converge to co-activate caspase-3 and induce apoptosis. Interestingly, curcumin might also inhibit the ERK signaling pathway, which blocks the cell cycle and down-regulates the expression of Bcl-2, simultaneously inhibiting the SHI-1 cell proliferation and inducing the apoptosis of these cells. In conclusion, three MAPK-related signal transduction pathways, driven by p-JNK, p-P38 MAPK, and p-ERK, may all be involved in curcumin-induced apoptosis of SHI-1 cells. The interaction of these three signaling molecules with both the intrinsic and extrinsic pathways induces SHI-1 cell apoptosis in a coordinated manner. However, the apoptosis-related genes p53 and survivin as well as the specific pathogenesis fusion gene MLL-AF6 may not be involved in this apoptotic process.
In addition, we also discovered that curcumin significantly reduces the mRNA expression of MMP-2 and MMP-9, reduces their enzymatic levels in cell supernatants, and partially suppresses the invasive ability of SHI-1 cells. MMPs are considered to be closely involved in tumor invasion and migration, and MMP-2 and MMP-9 belong to the gelatinase family of MMPs. Studies have confirmed that MMP-2 and MMP-9 play an important role in the invasion process of various solid tumors (Ray et al., Citation1994) and are also abnormally expressed in AML; their expression levels are correlated with extramedullary infiltration by leukemia cells (Reikvam et al., Citation2010). Feng reported that tight junction proteins were more obviously disrupted in SHI-1 cells than in the HL-60 and U937 leukemic cell lines, which is consistent with the higher rate of invasion of SHI-1 cells and the high constitutive expression of MMP-2 and MMP-9 in this cell line (Feng et al., Citation2011; Wang et al., Citation2010). MMP-2 and MMP-9 are tightly regulated at the levels of transcription and secretion by multiple factors. For example, various stimulating factors can trigger MAPK, NF-κB and PYC signaling pathways, leading to effects on the GC box in the MMP-2 promoter region or to effects on AP-1 and NF-κB binding to the MMP-9 promoter region for expression regulation (Gallera et al., Citation2015). Therefore, in this study, we further speculated that curcumin likely down-regulated the transcription and inhibited the secretion of MMP-2 and MMP-9 by triggering MAPK and NF-κB, which ultimately caused partial suppression of SHI-1 cell migration and invasion.
Conclusions
In this study, we discovered for the first time that curcumin not only significantly induces apoptosis in acute monocytic leukemia SHI-1 cells but also partially suppresses their invasion. To achieve this effect, curcumin likely activates MAPK-related pathways that are driven by JNK, P38 MAPK, and ERK, leading to increased FasL expression, reduced Bcl-2 expression, and NF-κB inhibition. Ultimately, this sequence of events leads to caspase-3 activation via both intrinsic and extrinsic apoptosis pathways, and SHI-1 cell apoptosis is subsequently induced. Furthermore, curcumin may also down-regulate the transcription of MMP-2 and MMP-9 and inhibit their activation by triggering MAPK and NF-κB, leading to partial suppression of SHI-1 cell migration and invasion. In the future, in vivo experiments should be performed to confirm the effect of curcumin on the apoptosis and invasion of human acute leukemia cells.
Declaration of interest
The authors report that they have no conflicts of interest. This work was supported by grants from the Natural Science Foundation of China (No. 81274139), Projects Supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 14KJB320010), and the Natural Science Foundation for the Youth of Nanjing University of Chinese Medicine (No. 13XZR13).
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